Great strides have been made over the past 60 years in reducing long-term mortality trends for some types of cancer, such as stomach cancer. However, during the same period, mortality trends for other cancers have remained stable or increased. For example, lung cancer is the most frequent cancer worldwide, representing the leading cause of cancer mortality among men and women. Breast cancer is the commonest cancer among women and the second leading cause of cancer mortality in women, and ovarian cancer mortality rates are increasing in some countries. Childhood and adult lymphatic cancers, such as leukemias and non-Hodgkin's lymphomas, also continue to represent significant causes of cancer mortality. Early diagnosis and effective treatment remains a goal for all of these cancers.
Several years ago, site directed diagnosis and therapy were proposed, to allow in vivo targeting of particular sites of disease within an animal's body. In general, site-directed diagnosis or therapy employs a targeting moiety, such as an antibody specific for the disease site or for the organism which caused the disease, coupled to a label in the case of a diagnostic agent or to a cytotoxic agent in the case of a therapeutic agent. A very large body of literature exists relating to radiolabeling antibodies or antibody fragments for diagnostic imaging purposes. Similarly, a number of site directed therapeutic agents employing monoclonal antibodies and a variety of radioisotopes have been proposed over the years, e.g., as set forth in U.S. Pat. Nos. 4,454,106; 4,472,509; 4,828,991; 5,246,691; 5,355,394; and 5,641,471; in EP 429624; EP 585986; WO 90/15625, and the like. Such antibody-based agents produce side effects related to the immune responses of the treated animal to the antibody, even if antibody fragments or humanized antibodies are employed as the targeting moiety.
The disadvantages of antibody-based site-directed diagnostic and therapeutic agents can be avoided when targeting moieties having lower molecular weights, such as receptor-specific peptides or small molecules are employed. However, coupling of a peptide or small molecule to a label or cytotoxic agent, while retaining the compound's receptor specificity, can be technically difficult. Methods for radiolabeling peptides and other small molecules which preserve the ability of the compound to bind specifically to a receptor are disclosed in commonly owned U.S. Pat. Nos. 5,225,180; 5,405,597; 5,443,815; 5,508,020; 5,552,525; 5,561,220; 5,620,675; 5,645,815; 5,654,272; 5,711,931; 5,716,596; 5,720,934; and 5,736,122; in abandoned U.S. patent application Ser. No. 07/955,466; and in WO92/13572, WO93/10747, WO93/17719, WO93/21962, WO93/23085, WO93/25244, WO94/00489, WO94/07918, and WO94/28942. The methods disclosed in these patents and publications are particularly suitable for manufacture of site-directed diagnostic imaging agents. Commonly assigned U.S. Pat. Nos. 5,620,675; 5,716,596; WO 94/00489; WO 95/03330; WO 95/00553; WO 95/31221; and WO 96/04308 disclose somatostatin peptide analogs which may be used for site-directed radiotherapy. Commonly assigned WO 95/33497 discloses somatostatin analogs, gpIIb/IIIa receptor-binding peptides, and leukocyte-binding peptides which may be used for site-directed radiodiagnosis or radiotherapy. Commonly assigned WO 96/30055 discloses vasoactive intestinal peptide (VIP) receptor-binding peptides which may be used for site-directed radiodiagnosis or radiotherapy.
Tumor cells often occasionally express or overexpress a particular receptor or receptor subtype, as indicated by receptor binding studies. In some types of cancer, tumor cell markers can change as the disease progresses, possibly reflecting the stage of the disease and thus the patient's prognosis. The kind of receptor that a tumor cell expresses can be characteristic of the tumor's etiology and can thus provide a relatively specific marker for the tumor. For example, radiolabeled somatostatin analogs have been shown to bind specifically to neuroendocrine tumors, melanomas, lung cancer, and certain breast cancers. One such analog, .sup.111 In-OCTREOSCAN, has received marketing approval for use in imaging neuroendocrine tumors. A second radiolabeled somatostatin analog, .sup.99m Tc-Depreotide, has completed Phase III clinical trials for use in imaging lung cancers. .sup.123 I-vasoactive intestinal peptide has been shown to target adenocarcinomas of the colon and stomach.
Calcitonin (CT) is a 32 amino acid peptide secreted from the thyroid in response to elevated serum calcium levels. Calcitonin has a number of biological effects, which are mediated by calcitonin receptors present on the surfaces of cells in the target organ. High affinity receptors for CT have been identified in bone, kidney, lung, and central nervous system. In bone, CT inhibits bone resorption by osteoclasts; in kidney, CT increases excretion of calcium ions; and in the central nervous system, the peptide induces analgesia, gastric acid secretion, and appetite inhibition. Small amounts of CT have been administered to animals and humans without toxic effects, and salmon CT is used clinically to treat such bone disorders as Paget's disease, hypercalcemia of malignancy, and osteoporosis. Intravenously-administered calcitonin clears the blood rapidly and is excreted primarily in urine. The major sites of localization for administered CT are kidney, liver and the epiphyses of the long bones.
Circulating CT levels are considered to be a marker for some types or stages of cancer, for example, medullary thyroid carcinoma, small-cell lung cancer, carcinoids, breast cancer, and gastrointestinal cancer. High affinity CT receptors have been identified in lymphoid cells, human lung cancer cell lines, human breast cancer cell lines, and in primary breast cancer tissue. Findlay et al. (1981) Biochem. J. 196: 513-520 reports that CT receptors are overexpressed in certain breast, lung, ovarian and lymphoma cancer cell lines.
The amino acid sequences of CT from several species (human, salmon and eel) are set forth below:
hCT CGNLSTCMLGTYTQDFNKFHTFPQTAIGVG.AP.amide (SEQ ID NO.: 1) PA0 sCT CSNLSTCVLGKLSQELHKLQTYPRTNTGSG.TP.amide (SEQ ID NO.:2) PA0 eCT CSNLSTCVLGKLSQELHKLQTYPRTDVGAGTP.amide (SEQ ID NO.:3) PA0 diethylenetriaminepentaacetic acid (DTPA) EQU (HOOCCH.sub.2).sub.2 N(CR.sub.2)(CR.sub.2)N(CH.sub.2 COOH)(CR.sub.2)(CR.sub.2)N(CH.sub.2 COOH) PA0 ethylenediaminetetraacetic acid (EDTA) EQU (HOOCCH.sub.2).sub.2 N(CR.sub.2)(CR.sub.2)N(CH.sub.2 COOH); PA0 1,4,7,10-tetraazadodecanetetraacetic acid; ##STR3## where n is an integer that is 2 or 3 and where each R is independently H, C.sub.1 to C.sub.4 alkyl, or aryl and one R is covalently linked to the CT receptor binding compound, and desferrioxamine. PA0 CH.sub.2 CO.SNLSTX-- (SEQ ID NO.:10) PA0 CH.sub.2 CO.X.sup.1 NLSTX.sup.2 --(SEQ ID NO.:11) PA0 CH.sub.2 CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGSGTP.amide; (SEQ ID NO.:5) PA0 CH.sub.2 CO.SNLST.Hcy.VLGKLSCELHKLQTYPRTNTGSGTP.amide; (SEQ ID No.:6) PA0 CH.sub.2 CO.SNLST.Cys.VLGKLSCELHKLQTYPRTNTGSGTP.amide; (SEQ ID NO.:7) and PA0 SNLST.Asu.VLGKLSCELHKLQTYPRTNTGSGTP.amide (SEQ ID NO.:8) PA0 CH.sub.2 CO.SNLST.Hhc.VLGKLSC(BAT)ELHKLQTYPRTNTGSGTP.amide (SEQ ID NO.:4) PA0 CH.sub.2 CO.SNLST.Hhc.VLGKLSQELHKLQTYPRTNTGSGTP(.epsilon.-K)GC.amide, PA0 CH.sub.2 CO.SNLST.Hhc.VLGKLSC(CH.sub.2 CO.GGCK.amide)ELHKLQTYPRTNTGSGTP.amide, PA0 CH.sub.2 CO.SNLST.Hhc.VLGKLSC(CH.sub.2 CO.(.beta.-Dap)KCK.amide)ELHKLQTYPRTNTGSGTP.amide, PA0 CH.sub.2 CO.SNLS T.Hhc.VLGKLSC(CH.sub.2 CO.(.epsilon.-K)GCE.amide)ELHKLQTYPRTNTGSGTP.amide, PA0 CH.sub.2 CO.SNLST.Hcv.VLGKLSC(CH.sub.2 CO.GGCK.amide)ELHKLQTYPRTNTGSGTP.amide, PA0 CH.sub.2 CO.SNLST.Hcy.VLGKLSC(CH.sub.2 CO.(.beta.-Dap)KCK.amide)ELHKLQTYPRTNTGSGTP.amide, PA0 CH.sub.2 CO.SNLST.Hcy.VLGKLSC(CH.sub.2 CO.(.epsilon.-K)GCE.amide)ELHKLQTYPRTNTGSGTP.amide PA0 CH.sub.2 CO.SNLST.Cys.VLGKLSC(C.sub.2 CO.GGCK.amide)ELHKLQTYPRTNTGSGTP.amide, PA0 CH.sub.2 CO.SNLST.Cys.VLGKLSC(CH.sub.2 CO.(.beta.-Dap)KCK.amide)ELHKLQTYPRTNTGSGTP.amide, PA0 CH.sub.2 CO.SNLST.Cys.VLGKLSC(CH.sub.2 CO.(.epsilon.-K)GCE.amide)ELHKLQTYPRTNTGSGTP.amide, PA0 SNLST.Asu.VLGKLSC(CH.sub.2 CO.(.beta.-Dap)KCK.amide)ELHKLQTYPRTNTGSGTP.amide, and PA0 SNLST.Asu.VLGKLSC(CH.sub.2 CO.(.beta.-Dap)KCK.amide)ELHKLQTYPRTDVGAGTP.amide. PA0 CH.sub.2 CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGSGTP.amide. (SEQ ID NO.:9)
(where single-letter abbreviations for amino acids can be found in Zubay, Biochemistry 2d ed., 1988, MacMillan Publishing: New York, p. 33, and where the underlined amino acids between the two cysteine residues in the amino terminal portion of the peptide represent a disulfide bond). Among species, nine residues, including the carboxyl-terminal proline amide and the disulfide-bonded cysteine residues at positions 1 and 7 are conserved. The salmon and eel CTs are more potent than human CT both in vitro and in vivo.
CT peptide analogs have been developed in which the chemically-labile disulfide is replaced with stable carbon-carbon linkages formed between 2-aminosuberic acid, as described in U.S. Pat. No. 4,086,221. CT analogs in which the amino terminal, midregion, or carboxyl terminal portion of the molecule are deleted demonstrate only weak binding to CT receptors. Many amino acid substitutions may be made between residues 8 and 22 of the CT molecule to generate biologically active CT analogs. Some CT analogs with only minimal sequence homology to any natural form of CT have biological activity similar to that of salmon CT. Truncated CT peptide derivatives (such as Cbz-LHKLQY-OMe) also retain substantial receptor binding activity.
Since tumors may express or overexpress different receptors, a variety of radiodiagnostic and radiotherapeutic agents are needed to afford optimal diagnostic and therapeutic modalities against cancer.